Generation Protocol of Human iPSCs from Peripheral Blood Using Lentiviral Vectors

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs). Patient-specific iPSCs lack the ethical concerns surrounding embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies.

Expansion of peripheral blood mononuclear cells (PBMCs)

• To start the protocol using frozen PBMCs, thaw 1 vial of cells into 10 ml of QBSF medium and centrifuge at 300×g for 10 min. Resuspend the cells in 2 ml of expansion medium (EM) and transfer them to one well of a 12-well plate. Incubate the cells in a 37°C, 5% CO₂ incubator.
• Transfer the cells to a sterile 15 ml conical tube and wash the well once with 1 ml of QBSF-60 stem cell medium to collect adherent cells. Centrifuge the cells at 300×g for 10 min.
• Resuspend the cells in 2 ml of EM and transfer to one well of a 12-well plate. Incubate the cells in a 37°C, 5% CO₂ incubator.

Transduction of PBMCs with lentivirus

• Resuspend the cells in 1 ml of fresh EM containing 5 μg/ml of polybrene and lentivirus and transfer to one well of a 12-well plate. Spin the plate at 2250 rpm at 25°C for 90 min.
• After spin, add 1 ml of fresh EM containing 5 μg/ml of polybrene for a total of 2 ml of medium and incubate the plate in a 37°C, 5% CO₂ incubator.
• The next day, resuspend the cells in 2 ml of EM and transfer to 1 well of a 12-well plate. Incubate the cells in a 37°C, 5% CO₂ incubator.

Plating transduced cells onto MEFs

• Coat the wells of a 6-well plate with 0.1% gelatin and plate-inactivated mouse embryonic fibroblasts (MEFs) at 2×10⁵ cells/well in MEF medium. Prepare 3 wells per infection.
• Resuspend the cells in 3 ml of MEF medium containing 10 ng/ml bFGF, ascorbic acid, and growth factors at the same concentrations as used in the EM medium.
• Plate 1 ml of cells per well of a 6-well plate containing MEFs. Add 1.5 ml of MEF media with bFGF, ascorbic acid, and growth factors for a total of 2.5 ml of media/well.
• Spin the plate at 500 rpm at 25°C for 30 min. Incubate the plate in a 37°C, 5% CO₂ incubator.
• Feed cells every other day with 2.5 ml of MEF media containing 10 ng/ml bFGF and 50 μg/ml ascorbic acid (no growth factors). Aspirate and discard floating cells with each feed.
• Once small colonies appear, feed cells daily with 2 ml of human embryonic stem cell (hESC) medium.

Picking and expansion of iPSC clones

• Pick each colony in individual wells of a 12-well plate pre-seeded with inactivated MEFs containing 1 ml/well of hESC medium plus 10 μM of ROCK inhibitor.

Creative Bioarray Relevant Recommendations

• Creative Bioarray is dedicated to providing several viable and cost-effective methods for pluripotent stem cell (iPSC) generation. We employ advanced iPSC reprogramming factor delivery by episomal vectors, viral vectors, as well as other iPSC reprogramming methods (mRNA, protein, and chemical reprogramming) to help you obtain the desired iPSC.

• When less than 5×10⁵ cells are transduced, cells may be plated onto one or two wells of a 6-well plate containing MEFs.
• We have experienced some variability in the time necessary to observe hESC-like colonies, in some cases up to 25 days post-transduction.

iPSC

iPSC Generation

Stem Cell Assay Development & Screening

For research use only. Not for any other purpose.